The goal of this high-risk/high-reward project is to develop the tools needed for experimental manipulation of human-associated methanogens. These commensal microorganisms colonize all humans, making significant contributions to host nutrition and overall health. The metabolic interactions between methanogens and other members of the human microbiota strongly influence the efficiency of the digestive process, as well as the colonic fermentation products that are absorbed by the host. Moreover, some human-associated methanogens are potent inducers of inflammatory cytokines, while others produce metabolites linked to the development of atherosclerosis and cancer. Given these observations, it is not surprising that methanogen carriage is correlated with a number of pathological conditions including obesity, inflammatory bowel disease, intestinal cancers and periodontal disease. Numerous hypotheses have been put forward to explain the linkage between methanogens and these health issues; unfortunately, experimental support for these ideas is exceedingly scarce. Three factors have contributed to this scarcity. First, all methanogens are exquisitely sensitive to oxygen and require specialized anaerobic techniques that are not widely employed; second, methods for genetic manipulation of human-associated methanogens are non-existent; and third, methanogen research has traditionally been restricted to the environmental and energy related topics. We propose to remedy this situation for the three of the dominant species of human-associated methanogens: Methanobrevibacter smithii, Methanospheara stadtmanae and the recently discovered Methanomassiliicoccus luminyensis. To achieve this goal we propose to develop genetic systems for human-associated methanogens and to acquire the genomic and transcriptomic resources needed to inform and direct the ongoing genetic analyses of these important microorganisms.